Historically, the detection and identification of bacteria, mycobacteria, yeasts, and molds have relied primarily on their morphologic and phenotypic properties. This approach is imprecise and slow for many clinically significant microbes. We have explored alternative methods, such as Sanger sequencing and mass spectrometry, for the detection and identification of selected organisms. Earlier studies from our laboratory demonstrated that Sanger sequencing and pyrosequencing were powerful tools for the definitive identification of bacteria including Nocardia and Mycobacterium, yeasts, and molds; however, these procedures are time-consuming and relatively expensive. Studies in the current fiscal year have focused on alternative identification methods, specifically MALDI-TOF (matrix-assisted laser desorption-ionization time of flight mass spectrometry) for the identification of Nocardia, mycobacteria, and molds and rapid genomic assays for identification of subspecies within the M. abscessus group. MALDI-TOF MS provides a reproducible spectral pattern based on the mass/charge (m/z) ratio of ionized proteins. Preparation of samples is rapid and inexpensive. Well-characterized reference strains of mycobacteria and molds allowed us to build a NIH database that was then challenged with clinical isolates. Following a demonstration of excellent performance, MALDI-TOF MS has been recently selected as the method of choice of the NIH Microbiology Service for identification of both rapid and slowly growing mycobacteria as well as filamentous molds and was also recently validated for use on Nocardia. Multi center evaluations of the performance of the NIH mold and Nocardia databases are ongoing.